Supramolecular Assembly and Thermogelation Strategies Using Peptide-Polymer Conjugates.

Peptide-polymer conjugates (PPCs) are of particular interest in the development of responsive, adaptive, and interactive materials due to the benefits offered by combining both building blocks and components. This review presents pioneering work as well as recent advances in the design of peptide-polymer conjugates, with a specific focus on their thermoresponsive behavior. This unique class of materials has shown great promise in the development of supramolecular structures with physicochemical properties that are modulated using soft and biorthogonal external stimuli. The temperature-induced self-assembly of PPCs into various supramolecular architectures, gelation processes, and tuning of accessible processing parameters to biologically relevant temperature windows are described. The discussion covers the chemical design of the conjugates, the supramolecular driving forces involved, and the mutual influence of the polymer and peptide segments. Additionally, some selected examples for potential biomedical applications of thermoresponsive PPCs in tissue engineering, delivery systems, tumor therapy, and biosensing are highlighted, as well as perspectives on future challenges.

Cucurbit[8]uril Mediated Supramolecular and Photocrosslinked Interpenetrating Network Hydrogel Matrices for 3D-Bioprinting.

Printing of biologically functional constructs is significant for applications in tissue engineering and regenerative medicine. Designing bioinks remains remarkably challenging due to the multifaceted requirements in terms of the physical, chemical, and biochemical properties of the three-dimensional matrix, such as cytocompatibility, printability, and shape fidelity. In order to promote matrix and materials stiffness, while not sacrificing stress relaxation mechanisms which support cell spreading, migration, and differentiation, this work reports an interpenetrating network (IPN) bioink design. The approach makes use of a chemically defined network, combining physical and chemical crosslinking units with a tunable composition and network density, as well as spatiotemporal control over post-assembly material stiffening. To this end, star-shaped poly(ethylene glycol)s functionalized with Phe-Gly-Gly tripeptide or photoactive stilbazolium are synthesized, and used to prepare three-dimensional networks with cucurbit[8]uril (CB[8]) through supramolecular host-guest complexation. The hydrogel obtained shows fast relaxation and thus supports the proliferation and differentiation of cells. Upon irradiation, the mechanical properties of the hydrogel can be rapidly adapted via selective photochemical dimerization of stilbazolium within CB[8], leading to IPNs with increased form stability while retaining the dynamic nature of the hydrogels. This modular approach opens new design opportunities for extrudable and cell-friendly dynamic biomaterials for applications in 3D-bioprinting.

Tuning the Cross-Linking Density and Cross-Linker in Core Cross-Linked Polymeric Micelles and Its Effects on the Particle Stability in Human Blood Plasma and Mice.

Core cross-linked polymeric micelles (CCPMs) are designed to improve the therapeutic profile of hydrophobic drugs, reduce or completely avoid protein corona formation, and offer prolonged circulation times, a prerequisite for passive or active targeting. In this study, we tuned the CCPM stability by using bifunctional or trifunctional cross-linkers and varying the cross-linkable polymer block length. For CCPMs, amphiphilic thiol-reactive polypept(o)ides of polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) [pSar-b-pCys(SO2Et)] were employed. While the pCys(SO2Et) chain lengths varied from Xn = 17 to 30, bivalent (derivatives of dihydrolipoic acid) and trivalent (sarcosine/cysteine pentapeptide) cross-linkers have been applied. Asymmetrical flow field-flow fraction (AF4) displayed the absence of aggregates in human plasma, yet for non-cross-linked PM and CCPMs cross-linked with dihydrolipoic acid at [pCys(SO2Et)]17, increasing the cross-linking density or the pCys(SO2Et) chain lengths led to stable CCPMs. Interestingly, circulation time and biodistribution in mice of non-cross-linked and bivalently cross-linked CCPMs are comparable, while the trivalent peptide cross-linkers enhance the circulation half-life from 11 to 19 h.

MUC1 Glycopeptide Vaccine Modified with a GalNAc Glycocluster Targets the Macrophage Galactose C-type Lectin on Dendritic Cells to Elicit an Improved Humoral Response.

Mucin expression and glycosylation patterns on cancer cells differ markedly from healthy cells. Mucin 1 (MUC1) is overexpressed in several solid tumors and presents high levels of aberrant, truncated O-glycans (e.g., Tn antigen). Dendritic cells (DCs) express lectins that bind to these tumor-associated carbohydrate antigens (TACAs) to modulate immune responses. Selectively targeting these receptors with synthetic TACAs is a promising strategy to develop anticancer vaccines and to overcome TACA tolerance. In this work, we prepared, via a solid phase peptide synthesis approach, a modular tripartite vaccine candidate, incorporating a high-affinity glycocluster based on a tetraphenylethylene scaffold, to target the macrophage galactose-type lectin (MGL) on antigen presenting cells. MGL is a C-type lectin receptor that binds Tn antigens and can route them to human leukocyte antigen class II or I, making it an attractive target for anticancer vaccines. Conjugation of the glycocluster to a library of MUC1 glycopeptides bearing the Tn antigen is shown to promote uptake and recognition of the TACA by DCs via MGL. In vivo testing revealed that immunization with the newly designed vaccine construct bearing the GalNAc glycocluster induced a higher titer of anti-Tn-MUC1 antibodies compared to the TACAs alone. Additionally, the antibodies obtained bind a library of tumor-associated saccharide structures on MUC1 and MUC1-positive breast cancer cells. Conjugation of a high-affinity ligand for MGL to tumor-associated MUC1 glycopeptide antigens has a synergistic impact on antibody production.

Impact of sample history and solvent effects on pathway control in the supramolecular polymerisation of Au(i)-metallopeptide amphiphiles.

We investigate the kinetics of the supramolecular polymerisation of an Au(i)-metallopeptide amphiphile that assembles into exceptionally long and rigid nanofibers. We developed a precise preparation protocol to measure the concentration dependent assembly kinetics which elucidated a nucleation-elongation dominated supramolecular polymerisation process. We show striking differences in the assembly behavior and morphology in aqueous media, even at organic solvent contents as low as 1 vol%, compared to pure buffer.

Can Polymer Helicity Affect Topological Chirality of Polymer Knots?

We investigate the effect of helicity in isolated polymers on the topological chirality of their knots with computer simulations. Polymers are described by generic worm-like chains (WLC), where helical conformations are promoted by chiral coupling between segments that are neighbors along the chain contour. The sign and magnitude of the coupling coefficient u determine the sense and strength of helicity. The corrugation of the helix is adjusted via the radius R of a spherical, hard excluded volume around each WLC segment. Open and compact helices are, respectively, obtained for R that is either zero or smaller than the length of the WLC bond, and R that is a few times larger than the bond length. We use a Monte Carlo algorithm to sample polymer conformations for different values of u, spanning the range from achiral polymers to chains with well-developed helices. Monitoring the average helix torsion and fluctuations of chiral order as a function of u, for two very different chain lengths, demonstrates that the coil-helix transition in this model is not a phase transition but a crossover. Statistical analysis of conformations forming the simplest chiral knots, 31, 51, and 52, demonstrates that topological mirror symmetry is broken─knots formed by helices with a given sense prefer one handedness over the other. For the 31 and 51 knots, positive helical sense favors positive handedness. Intriguingly, an opposite trend is observed for 52 knots, where positive helical sense promotes negative handedness. We argue that this special coupling between helicity and topological chirality stems from a generic mechanism: conformations where some of the knot crossings are found in "braids" formed by two tightly interwoven sections of the polymer.

Modular Platform of Carbohydrates-modified Supramolecular Polymers Based on Dendritic Peptide Scaffolds.

Glycopeptide supramolecular polymers displaying multivalent carbohydrates are particularly suitable for immune-relevant biomaterials, due to the important functions of carbohydrates in mediating cell-cell communication and modulating immune responses. However, the diversity and complexity of carbohydrates limited the generation of glycopeptide supramolecular monomers. Thereby, a modular platform of presenting various carbohydrates, especially more complex oligosaccharides, is highly desirable but remains underexplored. Here, we first prepared the linear amphiphilic glycopeptides that self-assembled into spherical nanoparticles and worm-like nanoparticles. Furthermore, the dendritic glycopeptides that self-assembled into uniform nanorods were designed to generate modular supramolecular polymers with variable functionality, via redesigning the molecular backbone. With various functional oligosaccharide-modified supramolecular polymers, the in vitro studies further indicated that these polymers were not cytotoxic to macrophages, and significantly modulated the production of proinflammatory cytokines. These findings provide a promising platform to develop supramolecular glycopeptide biomaterials with potential applications in immunomodulation and immunotherapy.

Construction of Active Protein Materials: Manipulation on Morphology of Salmon Calcitonin Assemblies with Enhanced Bone Regeneration Effect.

The effect of protein drugs is always limited by their relatively low stability and fast degradation property; thus, various elegant efforts have been made to improve the bioactivity and biocompatibility of the protein drugs. Here, an alternative way is proposed to solve this problem. By simply adding a limited amount of small-molecular regulator, which tunes the subtle balance of protein-protein interactions (PPIs) and disulfide bond formation, the self-assembly property of the protein drug can be regulated, forming an "active protein material" itself. This means that, the resulting biomaterial is dominated by the protein drug and water, with significantly enhanced bone regeneration effect compared to the virgin protein in vitro and in vivo, through multivalent effect between the protein and receptor and the retarded degradation of the assembled proteins. In this active protein material, the protein drug is not only the active drug, but also the drug carrier, which greatly increases the drug-loading efficiency of the biomaterial, indicating the advantages of the easy preparation, high efficiency, and low cost of the active protein material with a bright future in biomedical applications.

Bridging Rigidity and Flexibility: Modulation of Supramolecular Hydrogels by Metal Complexation.

The combination of complementary, noncovalent interactions is a key principle for the design of multistimuli responsive hydrogels. In this work, an amphiphilic peptide, supramacromolecular hydrogelator which combines metal-ligand coordination induced gelation and thermoresponsive toughening is reported. Following a modular approach, the incorporation of the triphenylalanine sequence FFF into a structural (C3 EG ) and a terpyridine-functionalized (C3 Tpy ) C3 -symmetric monomer enables their statistical copolymerization into self-assembled, 1D nanorods in water, as investigated by circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). In the presence of a terpyridine functionalized telechelic polyethylene glycol (PEG) cross-linker, complex formation upon addition of different transition metal ions (Fe2+ , Zn2+ , Ni2+ ) induces the formation of soft, reversible hydrogels at a solid weight content of 1 wt% as observed by linear shear rheology. The viscoelastic behavior of Fe2+ and Zn2+ cross-linked hydrogels are basically identical, while the most kinetically inert Ni2+ coordinative bond leads to significantly weaker hydrogels, suggesting that the most dynamic rather than the most thermodynamically stable interaction supports the formation of robust and responsive hydrogel materials.

The Development of Vaccines from Synthetic Tumor-Associated Mucin Glycopeptides and their Glycosylation-Dependent Immune Response.

Tumor-associated carbohydrate antigens are overexpressed as altered-self in most common epithelial cancers. Their glycosylation patterns differ from those of healthy cells, functioning as an ID for cancer cells. Scientists have been developing anti-cancer vaccines based on mucin glycopeptides, yet the interplay of delivery system, adjuvant and tumor associated MUC epitopes in the induced immune response is not well understood. The current state of the art suggests that the identity, abundancy and location of the glycans on the MUC backbone are all key parameters in the cellular and humoral response. This review shares lessons learned by us in over two decades of research in glycopeptide vaccines. By bridging synthetic chemistry and immunology, we discuss efforts in designing synthetic MUC1/4/16 vaccines and focus on the role of glycosylation patterns. We provide a brief introduction into the mechanisms of the immune system and aim to promote the development of cancer subunit vaccines.

Cell Adhesion on UV-Crosslinked Polyurethane Gels with Adjustable Mechanical Strength and Thermoresponsiveness.

Temperature-responsive polyurethane (PU) hydrogels represent a versatile material platform for modern tissue engineering and biomedical applications. However, besides intrinsic advantages such as high mechanical strength and a hydrolysable backbone composition, plain PU materials are generally lacking bio-adhesive properties. To overcome this shortcoming, the authors focus on the synthesis of thermoresponsive PU hydrogels with variable mechanical and cell adhesive properties obtained from linear precursor PUs based on poly(ethylene glycol)s (pEG) with different molar masses, isophorone diisocyanate, and a dimerizable dimethylmaleimide (DMMI)-diol. The cloud point temperatures of the dilute, aqueous PU solutions depend linearly on the amphiphilic balance. Rheological gelation experiments under UV-irradiation reveal the dependence of the gelation time on photosensitizer concentration and light intensity, while the finally obtained gel strength is determined by the polymer concentration and spacing of the crosslinks. The swelling ratios of these soft hydrogels show significant changes between 5 and 40 °C whereby the extent of this switch increases with the hydrophobicity of the precursor. Moreover, it is shown that the incorporation of a low amount of catechol groups into the networks through the DMMI dimerization reaction leads to strongly improved cell adhesive properties without significantly weakening the gels.

Magnetic Control over the Fractal Dimension of Supramolecular Rod Networks.

Controlling supramolecular polymerization is of fundamental importance to create advanced materials and devices. Here we show that the thermodynamic equilibrium of Gd3+-bearing supramolecular rod networks is shifted reversibly at room temperature in a static magnetic field of up to 2 T. Our approach opens opportunities to control the structure formation of other supramolecular or coordination polymers that contain paramagnetic ions.

Facile access to foldable redox-active flavin-peptide conjugates.

A convenient approach for the synthesis of foldable redox-active flavin peptide conjugates was established. A model ß-hairpin oligopeptide motif was utilized to demonstrate that azidolysine side-chains are readily functionalised with an alkyne-bearing flavine derivative. The folding equilibrium of the peptide backbone as well as the redox behaviour of the flavin moieties remains intact after the conjugation.

Targeted Repolarization of Tumor-Associated Macrophages via Imidazoquinoline-Linked Nanobodies.

Tumor-associated macrophages (TAMs) promote the immune suppressive microenvironment inside tumors and are, therefore, considered as a promising target for the next generation of cancer immunotherapies. To repolarize their phenotype into a tumoricidal state, the Toll-like receptor 7/8 agonist imidazoquinoline IMDQ is site-specifically and quantitatively coupled to single chain antibody fragments, so-called nanobodies, targeting the macrophage mannose receptor (MMR) on TAMs. Intravenous injection of these conjugates result in a tumor- and cell-specific delivery of IMDQ into MMRhigh TAMs, causing a significant decline in tumor growth. This is accompanied by a repolarization of TAMs towards a pro-inflammatory phenotype and an increase in anti-tumor T cell responses. Therefore, the therapeutic benefit of such nanobody-drug conjugates may pave the road towards effective macrophage re-educating cancer immunotherapies.

Supramolecular polymerization of sulfated dendritic peptide amphiphiles into multivalent L-selectin binders.

The synthesis of a sulfate-modified dendritic peptide amphiphile and its self-assembly into one-dimensional rod-like architectures in aqueous medium is reported. The influence of the ionic strength on the supramolecular polymerization was probed via circular dichroism spectroscopy and cryogenic transmission electron microscopy. Physiological salt concentrations efficiently screen the charges of the dendritic building block equipped with eight sulfate groups and trigger the formation of rigid supramolecular polymers. Since multivalent sulfated supramolecular structures mimic naturally occurring L-selectin ligands, the corresponding affinity was evaluated using a competitive SPR binding assay and benchmarked to an ethylene glycol-decorated supramolecular polymer.

Defects and defect engineering in Soft Matter.

Soft matter covers a wide range of materials based on linear or branched polymers, gels and rubbers, amphiphilic (macro)molecules, colloids, and self-assembled structures. These materials have applications in various industries, all highly important for our daily life, and they control all biological functions; therefore, controlling and tailoring their properties is crucial. One way to approach this target is defect engineering, which aims to control defects in the material's structure, and/or to purposely add defects into it to trigger specific functions. While this approach has been a striking success story in crystalline inorganic hard matter, both for mechanical and electronic properties, and has also been applied to organic hard materials, defect engineering is rarely used in soft matter design. In this review, we present a survey on investigations on defects and/or defect engineering in nine classes of soft matter composed of liquid crystals, colloids, linear polymers with moderate degree of branching, hyperbranched polymers and dendrimers, conjugated polymers, polymeric networks, self-assembled amphiphiles and proteins, block copolymers and supramolecular polymers. This overview proposes a promising role of this approach for tuning the properties of soft matter.

Lipid Bilayer Interactions of Peptidic Supramolecular Polymers and Their Impact on Membrane Permeability and Stability.

The synthesis and physicochemical characterization of supramolecular polymers with tunable assembly profiles offer exciting opportunities, involving the development of new biomedical carriers. Because synthetic nanocarriers aim to transport substances across or toward cellular membranes, we evaluated the interactions of amphiphilic peptide-based supramolecular polymers with lipid bilayers. Here, we focused on nanorod-like supramolecular polymers, obtained from two C3-symmetric dendritic peptide amphiphiles with alternating Phe/His sequences, equipped with a peripheral tetraethylene glycol dendron (C3-PH) or charged ethylenediamine end groups (C3-PH+). Triggered by pH changes, these amphiphiles assemble reversibly. Our results show that the supramolecular polymers have an impact on the lipid order in model membranes. Changes in the lipid order were observed depending on the charge state of the amphiphilic building blocks, as well as the chemical composition and physical properties of the bilayer. Furthermore, we further performed cell viability assays with the C3-PH+ and C3-PH supramolecular polymers. For C3-PH, the cell viability and extent of proliferation were decreased and the membrane permeability was enhanced, indicating a strong interaction of the polymer with cellular membranes. The results have implications for the design of novel pH-switchable supramolecular drug carriers and delivery vehicles that can respond to an altered microenvironment of tumorous or inflamed tissue.

Impact of NDI-Core Substitution on the pH-Responsive Nature of Peptide-Tethered Luminescent Supramolecular Polymers.

The pH-responsive nature of two self-assembled NDI-peptide amphiphile conjugates is reported. The diethoxy substituted NDI showed a pH-dependent assembly behaviour, as expected. In contrast, the isopropylamino- and ethoxy-substituted NDI based supramolecular polymer was stable at acidic and basic aqueous conditions. This finding highlights how subtle changes in the molecular design of π-stacked chromophore-peptide conjugates have a drastic impact on their equilibrium structure and ultimately functional properties.

Evaluation of Charge-Regulated Supramolecular Copolymerization to Tune the Time Scale for Oxidative Disassembly of ß-Sheet Comonomers.

A multistimuli-responsive supramolecular copolymerization is reported. The copolymerization is driven by hydrogen bond encoded ß-sheet-based charge co-assembly into 1D nanorods in water, using glutamic acid or lysine residues in either of the peptide comonomers. The incorporation of methionine as hydrophobic amino acid supports ß-sheet formation, but oxidation of the thioether side-chain to a sulfoxide functional group destabilizes the ß-sheet ordered domains and induces disassembly of the supramolecular polymers. Using H2 O2 as reactive oxygen species, the time scale and kinetics of the oxidative disassembly are probed. Compared to the charge neutral homopolymers, it is found that the oxidative disassembly of the charged ampholytic copolymers is up to two times faster and is operative at neutral pH. The strategy is therefore an important addition to the growing field of amphiphilic polythioether containing (macro)molecular building blocks, particularly in view of tuning their oxidation induced disassembly which tends to be notoriously slow and requires high concentrations of reactive oxygen species or acidic reaction media.